Project Summary/Abstract Chlamydia is one of the most important infectious agents from a public health perspective. More than 1.5 million cases of chlamydial infections are reported to the CDC annually, making it the most commonly reported infectious disease in the U.S. Chlamydia causes an unusual intracellular infection in which there is conversion between two developmental forms of the bacterium: the elementary body (EB), which is the infectious but dormant form, and the reticulate body (RB), which is the intracellular, replicating form. Gene expression is limited to RBs, and transcriptional silencing in EBs has been attributed to DNA condensation by two histone-like proteins, HctA and HctB. We hypothesize a second silencing mechanism in which RNA polymerase in EBs is inhibited in a reversible manner that is controlled by Type 3 secretion (T3S) activity. In Aim 1 we will study how a T3S chaperone Scc4, together with two other T3S proteins, coordinately regulates RNA polymerase activity and T3S secretion activity. In Aim 2, we will investigate a second T3S chaperone Scc2 that we propose as a T3S-regulated silencer of chlamydial transcription. In Aim 3, we will study the histone-like proteins to determine if they silence transcription uniformly or in a promoter-specific manner. These studies will define the mechanisms of transcriptional silencing in EBs, which is a central feature of developmental gene regulation in the intracellular Chlamydia infection and a potential therapeutic target.